Angiogenesis, the sprouting of new blood vessels from existing vasculature, is a critical step in the progression and metastasis of solid tumors. The long-term goal of our research is to dissect the molecular mechanisms of tumor angiogenesis. The purpose of the proposed research is to investigate how the Eph family of receptor tyrosine kinases regulate the growth of new blood vessels in tumors. Receptor tyrosine kinases represent a major class of cell-surface molecules that regulate tumor neovascularization. Members of Eph receptors and ligands are required in embryonic vascular development, yet their functions in postnatal angiogenesis remains unclear. Our preliminary results now show that ephrinA1 ligand and EphA2 receptor are expressed in two types of murine tumors and associated vasculature. A soluble EphA2 receptor abrogates islet tumor neovascularization in a vascular window assay, inhibits the formation of angiogenic islets in RIP-Tag transgenic mice, and suppresses 4T1 breast cancer growth and angiogenesis in vivo. To dissect the mechanisms of EphA receptor-mediated angiogenesis, we found that VEGF induces ephrinA1 expression and phosphorylation of EphA2 receptor in endothelial cells, and blocking EphA receptor activation inhibits VEGF-, but not FGF-induced endothelial cell survival, migration, sprouting, and corneal angiogenesis. Taken together, these data support a hypothesis that EphA RTKs and their ligands, ephrinAs, play a critical role in tumor neovascularization, and actions of ephrinA lignads on endothelial cells are required, at least in part, for VEGF-induced angiogenesis. To test this hypothesis, we will (1) determine whether activation of EphA2 receptor mediates VEGF-induced endothelial responses, (2) dissect the mechanisms of interactions between VEGF and the EphA/ephrinA signaling in angiogenesis, and (3) investigate whether EphA receptor activation is necessary and sufficient to promote tumor growth and angiogenesis in vivo using RIP-Tag transgenic tumor model. Each of these specific aims represents a different, but complementary, approach to understand the role of Eph receptor tyrosine kinases in tumor angiogenesis. These studies at the molecular, cellular, and whole animal level will make significant advances to the understanding of how different angiogenic factors are coordinated to promote tumor angiogenesis, and may identify novel targets to inhibit neovascularization in cancer.